Cancer prevention involving reduction or elimination of human exposure to environmental carcinogens may not always be possible. Inhibition of the development of cancer by the administration of anticarcinogenic agents may offer practical alternatives for reducing human cancer burden. However, the successful utilization of chemopreventive interventions will require solid mechanistic understanding of the action(s) of these agents. We have identified the Keap1-Nrf2-ARE signaling pathway as a target for chemoprevention. The Nrf2 transcription factor regulates an integrated cell survival response that can be triggered by multiple classes of cancer chemopreventive agents (e.g., dithiolethiones, isothiocyanates, triterpenoids). In this project, we will use both pharmacologic and genetic approaches to further probe the molecular mechanisms of action of these chemopreventive agents, assess their efficacy in animal models with close relevance to human carcinogenesis and use these interventions to validate intermediate biomarkers. With a continuing focus on the roles of infection with hepatitis B virus and co-exposure to the fungal toxin aflatoxin on human risk for hepatocellular carcinoma, we will purse three aims. First, we will utilize advanced methods of isotope-dilution mass spectrometry for the quantitation of aflatoxin biomarkers in liver, blood, urine and feces in order to develop for the first time a comprehensive mass balance for the fate of the ultimate carcinogen, exo-aflatoxin-epoxide, in rats. The predictive value of these biomarkers for individual risk of liver cancer will be assessed. Biomarkers will be quantified longitudinally during a bioassay for protection against hepatocarcinogenesis by the exceptionally potent triterpenoid activator of Nrf2 signaling, CDDO-Im. We will also evaluate the predictive value of monitoring DNA circulating in plasma for mutations in target oncogenes using quantitative short oligonucleotide mass spectrometry. In the second aim, we will evaluate the similarities and distinctions of chemical class, species and genetic activation of Keap1-Nrf2 signaling by comparing the gene expression patterns in rat and mouse liver following treatment with lead compounds of 3 different chemical classes of Nrf2 activators at doses equi-effective for inhibition of aflatoxin-induced preneoplastic lesions and by comparing the gene expression patterns in liver of mice in which either Nrf2 or its repressor Keap1 have been genetically disrupted. The impact of these genetic and pharmacologic interventions on aflatoxin disposition will be determined using the mass spectrometry-based analysis of its biomarkers. Third, we will capitalize on our novel observation that Nrf2 signaling influences tissue regeneration and repair by examining the post initiation effects of triterpenoids during aflatoxin-induced hepatocarcinogenesis in the rat and in a murine model recapitulating major human risk factors for HCC, namely, aflatoxin exposure and infection with HBV. Collectively, these studies will further the goal of effectively using activators of Nrf2 signaling as protective agents in human populations exposed to environmental toxicants. PUBLIC HEALTH RELEVANCE: Cancer prevention involving reduction or elimination of human exposure to environmental carcinogens may not always be possible. The overriding goal of our work remains to provide the mechanistic framework to facilitate the efficient translation of the most effective of the small molecule activators of Nrf2 signaling into use as protective agents in human populations exposed to environmental toxicants such as aflatoxins. To facilitate this goal we need better understanding of the molecular mechanisms of action of our chemopreventive agents, further validation of intermediate biomarkers, and assessment of efficacy in animal models with close relevance to human carcinogenesis.